Abstract
Kinetic studies of the early events in cytochrome c folding are reviewed with a focus on the evidence for folding intermediates on the submillisecond timescale. Evidence from time-resolved absorption, circular dichroism, magnetic circular dichroism, fluorescence energy and electron transfer, small-angle X-ray scattering and amide hydrogen exchange studies on the t ≤ 1 ms timescale reveals a picture of cytochrome c folding that starts with the ~ 1-μs conformational diffusion dynamics of the unfolded chains. A fractional population of the unfolded chains collapses on the 1 – 100 μs timescale to a compact intermediate IC containing some native-like secondary structure. Although the existence and nature of IC as a discrete folding intermediate remains controversial, there is extensive high time-resolution kinetic evidence for the rapid formation of IC as a true intermediate, i.e., a metastable state separated from the unfolded state by a discrete free energy barrier. Final folding to the native state takes place on millisecond and longer timescales, depending on the presence of kinetic traps such as heme misligation and proline mis-isomerization. The high folding rates observed in equilibrium molten globule models suggest that IC may be a productive folding intermediate. Whether it is an obligatory step on the pathway to the high free energy barrier associated with millisecond timescale folding to the native state, however, remains to be determined.
Highlights
The small globular protein cytochrome c has played a large role in studies of the earliest events and intermediates in protein folding
A more controversial corollary of this scenario is the proposal that no other kinetic folding intermediates exist between U, the fully denatured state, and the free energy barrier associated by the hydrogen exchange (HX) evidence with N-/C-terminal helix formation [19]
~ kBT of the unfolded state at low denaturant concentrations [53,22]. This suggests that IC probably does not correspond to the M state observed in equilibrium guanidine hydrochloride (GuHCl) unfolding studies, as the latter state appears to be more stable at zero denaturant
Summary
The small globular protein cytochrome c has played a large role in studies of the earliest events and intermediates in protein folding. The rate-limiting step in this folding pathway is either the formation of the highest energy foldon (N-/C-terminal helices) or, in the presence of an additional barrier from heme misligation, a later metastable intermediate, e.g., the formation of the 60’s helix and residue 25 – 30 loop This placement of the largest activation barrier at or near the beginning of the pathway is required by the fact that sequentially folded intermediates like those just described have remained largely hidden from observation in cyt c. A more controversial corollary of this scenario is the proposal that no other kinetic folding intermediates exist between U, the fully denatured state, and the free energy barrier associated by the HX evidence with N-/C-terminal helix formation [19] This view implies that the timescales for cooperative molecular collapse to a state with nearnative compaction and for formation of elements of native secondary structure can proceed no faster than ~ 1 – 10 ms in cyt c. This contention is discussed further below after considering the experimental evidence for molecular collapse and helix formation processes on the microsecond timescale
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